1. Technical Field
The present invention relates to recombinant DNA technology and more specifically towards genetic screens for identifying protein activities.
2. Background Art
The use of forward genetics has led to the identification of gene products involved in numerous processes from retroviral infection to signal transduction to oncogenesis (Deng, H. K., et al., Lund, A. H., et al., Walting, D., et al., and Zhong, Z., et al.). The power of these approaches rests in the virtual exclusion of experimenter bias and the breadth of candidate genes that can be tested in a single experiment. Every forward genetics technique, however, possesses intrinsic advantages and limitations (Stark, G. R., et al.). For instance, retrovirus-based insertional mutagenesis has the advantage of nearly random proviral insertion, which results in relatively normalized gene targeting. This allows one to reveal gain-of-function effects through gene activation even for rarely expressed or normally tissue-specific gene products. Because most eukaryotic cell lines are polyploid, however, repeated targeting of a single locus is required for loss-of-function effects (Harrington, J. J., et al.). By comparison, cDNA library-based expression cloning can uncover both gain-of-function and loss-of-function effects; although, it is often limited by unequal gene representation within the cDNA library (Simonsen, H., et al.). Because the strength of one experimental approach is often the defect of another, two experimental approaches can be combined towards a synergistic gain in screening power. For example, retrovirus-based insertional mutagenesis and cDNA expression cloning can be combined in a screen that aims to identify a specific protein activity.
Cytokines are a unique family of growth factors. Secreted primarily from leukocytes, cytokines stimulate both humoral and cellular immune responses, as well as the activation of phagocytic cells. One type of cytokine is the transforming growth factor βs (TGFβ). TGFβ was originally characterized as a protein (secreted from a tumor cell line) that was capable of inducing a transformed phenotype in non-neoplastic cells in culture. This effect was reversible, as demonstrated by the reversion of the cells to a normal phenotype following removal of the TGFβ. Subsequently, many proteins homologous to TGFβ have been identified. The four closest relatives are TGFβ1 (the original TGFβ) through TGFβ5 (TGFβ1 through TGFβ4). All four of these proteins share similar amino acid regions.
The TGFβ related family of proteins includes the activin and inhibin proteins. There are activin A, B, and AB proteins, as well as an inhibin A and inhibin B protein. The Mullerian inhibiting substance (MIS) is also a TGFβ related protein, as are members of the bone morphogenetic protein (BMP) family of bone growth-regulatory factors. Indeed, the TGFβ family can comprise as many as 100 distinct-proteins, all with at least one region of amino-acid sequence homology.
TGFβs have proliferative effects on many mesenchymal and epithelial cell types. Under certain conditions TGFβ demonstrates anti-proliferative effects on endothelial cells, macrophages, and T- and B-lymphocytes. Such effects include decreasing the secretion of immunoglobulin and suppressing hematopoiesis, myogenesis, adipogenesis, and adrenal steroidogenesis. Several members of the TGFβ family are potent inducers of mesodermal differentiation in early embryos, in particular TGF-β and activin A.
TGFβs, specifically TGFβ1, 2, and 3, are multipotent cytokines that are important modulators of cell growth, inflammation, matrix synthesis, the immune system, angiogenesis, and apoptosis (Taipale, J., et al.). Defects in TGFβ function are associated with a number of pathological states including immunosuppression, tumor cell growth, fibrosis, and autoimmune disease (Blobe, et al.). The TGF-βs are the prototypes of the TGF-β superfamily that consists of over 40 members that control key events in early development, patterning, tissue repair and wound healing. Although much is known about the regulation of TGF-β expression and signaling, the control of extracellular TGF-β availability is poorly understood. This is important because TGF-β is released as part of a latent complex in which the cytokine cannot interact with its receptor.
For TGF-β to signal, it must be released from its inactive complex by a process called activation. The latent complex consists of the 25 kD TGF-β homodimer, the TGF-β propeptides, also called the latency associated protein (LAP), and the latent TGF-β binding protein (LTBP). Even though the bond between the TGF-βs and LAP is cleaved within the Golgi, the TGF-β propeptide remains bound to TGF-β by non-covalent interactions. The complex of TGF-β and LAP is called the small latent complex (SLC). It is the association of LAP and TGF-β that confers latency. LAP-TGF-β binding is reversible and the isolated purified components can recombine to form an inactive SLC.
An important consideration for TGF-β action is the difference between the terms “activation” and “processing.” For the TGF-βs, the term “processing” refers to the proteolytic cleavage of the bond between TGF-β and LAP. Without cleavage, no TGF-β activity can be detected in the precursor TGF-β dimer under any conditions. Cleavage is a prerequisite for activity. The term “activation” refers to the liberation of the TGF-β dimer from its interaction with LAP. Therefore, the “processed” TGF-β precursor has the potential to be activated, i.e. to release TGF-β, whereas the unprocessed TGF-β cannot be activated without initially cleaving (processing) the propeptide bond.
Several molecules have been described as latent TGF-β activators. The first cell-mediated activation process in which several cell types converted the LLC, which is produced constitutively by most cells, into active TGF-β by a protease-dependant reaction was by proteases. Latent TGF-β activation required a) the protease urokinase plasminogen activator (uPA), b) activation of uPA's substrate plasminogen (the zymogen of the protease plasmin), c) binding of LAP to cell surface mannose-6 (M6P) phosphate/IGF-II receptors, d) LTBP, and e) TGase, as antibodies and/or inhibitors of each of these reactants blocked latent TGF-β activation. A number of other proteases, including MMP-2, MMP-9, plasmin, calpain, chymase, and elastase have subsequently been described as latent TGF-β activators (Koli, et al. (2001)).
A second mechanism for latent TGF-β activation involves the interaction of the matricellular protein thrombospondin (TSP-1) with latent TGF-β in a multi-molecular complex containing TSP-1 receptors as well as CD36, and, in some cases, plasmin. Latent TGF-β activation involves a direct interaction between TSP-1 and LAP and includes the tripeptide sequence RFK found in the TSP-1 type 1 repeats. This peptide is believed to interact with the conserved tetra peptide LSKL in the LAP amino terminus disrupting the non-covalent association between LAP and TGF-β. A tetra peptide KRFK will activate latent TGF-β in vitro and in vivo, whereas addition of the LAP peptide (LSKL) in excess blocks latent TGF-β activation. TSP-1−/− mice show a partial, overlapping phenotype with TGF-β1−/− mice with respect to enhanced inflammation. The administration of the LSKL blocking peptide to wild type mice induces pancreas and lung pathologies similar to those observed in TGF-β−/− animals, whereas the addition of the KRFK activating peptide to TSP-1−/− mice reverts the phenotype towards normal. However, the phenotype of the TSP-1−/− mouse does not replicate the full phenotype of the TGF-β1−/− mouse nor does the TSP-1−/− phenotype resemble any of the phenotypes of the TGF-β2−/− or TGF-β3−/− mice. These discrepancies again suggest that there may be multiple and isoform specific mechanisms for activation of latent TGF-β.
Latent TGF-β can be activated by mild acid (pH 4.5), which probably destabilizes the interaction between LAP and TGF-β. However, except for specialized situations, such as the extracellular compartment formed by osteoclasts during bone resorption, this pH is probably rarely achieved in the extracellular environment in vivo. Therefore, pH is unlikely to be a common mechanism for TGF-β activation.
The TGF-β1 and β3, but not TGF-β2, propeptides contain the integrin recognition sequence RGD. TGF-β1 and TGF-β3 LAPs interact with cells expressing the integrins αvβ1 and αvβ5. Although the binding of latent TGF-β with these integrins does not result in activation, the ligation of latent TGF-β with αvβ6 results in activation (J. S. Munger, et al (1999)). Activation of latent TGF-β1 or β3 by αvβ6 requires the RGD sequence as mutant forms of TGF-β1 or β3 containing RGE fail to be activated. The integrin, αvβ8, in combination with MT1-MMP, activates latent TGF-β (D. Mu, et al, (2002)). The expression of integrin αvβ6 is restricted to epithelia, and under normal conditions β6 expression is low. However, during inflammation, β6 expression is enhanced dramatically (αv expression is constitutively high in most cells.) Because TGF-β is a powerful suppressor of inflammation, the heightened expression of β6, and subsequent activation of latent TGF-β, provides a potent mechanism for the down-modulation of the inflammatory state.
The ability of β6 integrin to activate latent TGF-β and the known profibrotic effects of TGF-β (W. A. Border, et al. (1992)) are illustrated in vivo by the fact that mice develop pulmonary inflammation followed by fibrosis in response to the inflammatory and profibrotic drug bleomycin. Because TGF-β enhances the expression of β6 by alveolar cells, bleomycin probably initiates a feed-forward mechanism by coordinately upregulating both integrin expression and TGF-β generation. In this scenario, fibrosis is the result of a failure to interrupt this loop. Interestingly, β6−/− mice have only a minor fibrotic response to bleomycin (J. S. Munger, et al. (1998)). However, although β6−/− mice display certain overlapping phenotypes with TGF-β1−/− mice, β6−/− mice do not phenocopy all aspects of TGF-β−/− mice. Moreover, there is no overlap of β6−/− and TGF-β3−/− phenotypes indicating that additional mechanisms exist for TGF-β3 generation.
Whereas the known mechanisms for latent TGF-β activation may account for the TGF-β observed in some situations, none of the established mechanisms accounts for all activation reactions; nor do these processes account for all of the TGF-β null phenotypes. Moreover, there are reports of active TGF-β production under conditions where the known mechanisms do not seem to apply. Certain glioblastoma or myeloma cell lines release active TGF-β, but this process cannot be blocked by inhibitors of TSP-1, proteases or αvβ6. These results indicate that activation mechanisms in addition to those already described exist.
An unusual property of TGFβ is that its activity is limited by the conversion of latent TGFβ to active TGFβ (a process termed latent TGFβ activation). Tissues contain significant quantities of latent TGFβ and activation of only a small fraction of this latent TGFβ generates maximal cellular responses (Annes, et al. (2003)). Latency is conferred by the non-covalent interaction of the TGFβ propeptide, also called the latency associated protein (LAP), with the mature cytokine after cleavage of the bond between TGFβ and LAP has occurred (Annes, et al.). Once active, TGFβ binds and brings together its high affinity serine/threonine kinase type I and type II receptors and initiates a signal transduction cascade (Massague, et al.). Although genetic screens have identified molecules involved in TGFβ signaling (Hocevar, B. A., et al., Rodriguez, C., et al., and Sun, P., et al.), no screen as of yet identifies genes involved in the process of latent TGFβ activation.
A number of gene products are considered to be latent TGF-β activators, including the integrins ανβ6 (Munger, J. S., et al. and Annes, J. P., et al. (2002)) and ανβ8 (Mu, D., et al.), plasmin (Lyons, R. M., et al.), thrombospondin-I (Yu, Q., et al.), matrix metalloproteinases (Yu, Q., et al.), and others (Annes, J. P., et al., (2003)). However, there is limited in vivo supporting evidence that these molecules are latent TGFβ activators. In fact, the phenotypes of mice with null mutations in TGFβ1, 2, and 3 genes can only be partially accounted for by the currently identified latent TGFβ activating molecules (Kulkarni, A. B., et al., Sanford, L. P., et al., and Kaartinen, V., et al.). Furthermore, TGFβ is generated by unknown mechanisms by: (1) certain cell lines (Fernandez, T., et al., Horimoto, M., et al., and Olofsson, A., et al.); (2) cells treated with various compounds (retinoids (Glick, A. B., et al.), antiestrogens (Knabbe, C., et al.), vitamin D3 (Koli, K., et al.), glucocorticoids (Oursler, M. J., et al., and Boulanger, J., et al.); and (3) disease states (Border, W. A., et al., and Blobe, G. C., et al.).
Accordingly, there is a need for a method and screen that focuses on identifying the extracellular regulation or activity of bioactive signaling molecules. More specifically, there is a need for identifying latent TGF-β activators and the mechanisms of extracellular regulation of TGF-β.